The present invention relates to materials and methods which may be used in the detection and manipulation of Pre-Harvest Sprouting (PHS) and other dormancy-related phenotypes in plants. The present invention also relates to materials and methods for use in plant breedingxe2x80x94in particular to molecular-biology based methods for generating, identifying, characterising or manipulating genetic variation which affects the PHS and other dormancy related traits.
Pre-Harvest Sprouting (PHS) of non-dormant grains is a major limiting factor in achieving consistent bread making quality of UK wheat. Average annual losses due to PHS in the UK wheat crop have been estimated at some £17 million, but the problem is erratic and is much more severe in cool, damp seasons. Variation in the degree of sprouting damage from year to year makes this problem difficult to select against in conventional breeding programmes.
Previous work in other plant species has shown that the VIVIPAROUS 1 gene is a major regulator of embryo maturation in maize. Thus McCarty et al (1989) in The Plant Cell 1, 523-532 disclosed that vp1 mutants in maize were abscisic acid (ABA) insensitive, and demonstrated its role in controlling the developmental responses associated with the maturation phase of seed formation. VP1 mutants were shown to germinate precociously. Similarly McCarty et al (1991) in Cell 55, 895-905 disclosed that VP1 encoded 73 kDa transcription factor.
Giraudat et al (1992) in The Plant Cell 4, 1251-1261 showed that Arabidopsis ABI3 mutants had altered seed development and germination. The predicted gene product was similar to VP1 protein. These results and others show that ABI 3/VP1 function as developmental regulators during the maturation stage of embryogenesis by regulating transcription of sets of genes that determine the embryonic phenotype in preparation for desiccation of the seed prior to shedding.
A VP1 homolog in rice has also been isolated (Hattori et al (1994) Plant Molecular Biology 24, 805-810). Similarly in Abstract, Poster No P184 xe2x80x9cPoaceae sequence analysis: cloning of a VP-1 homolog from genomic barley DNA via PCRxe2x80x9d, at the Plant and Animal Genome V Conference in San Diego, USA, January 1997, Wilson and Sorrells disclosed the use of conserved primers to pick out vp1 homologs in Barley.
Wilson speculated that a wheat VP1 homolog may be obtainable by comparison with maize, rice and barley sequences, and (on the basis of comparison with these species) that the R locus may contain a wheat VP1 homolog.
Interestingly, earlier work by Cadle et al (1994) in Genome 37, 129-132 had already shown that the maize vp1 from McCarty didn""t hybridise strongly to wheat DNA and could not therefore be used as a probe to map the wheat gene, although various ABA-inducible genes were mapped successfully.
Other recent studies of the genetics of the transition from embryogenesis to germination in maize and Arabidopsis show that mutation of GA (gibberellic acid) and ABA synthesis and sensitivity can alter dormancy levels (Koornneef and Karssen, 1994). For example, whereas the Arabidopsis mutation ga 1 causes a loss of germination due to GA deficiency, aba/abi mutations (that affect ABA synthesis and perception respectively) cause a loss of dormancy (and in strong alleles, loss of viability) because embryos fail to develop desiccation tolerance during maturation (for example alleles of abi 3, Ooms et al., 1993).
It has been suggested that PHS in wheat is the result of the lack of induction of dormancy during embryo development (Gale and Lenton 1987). As is known to those skilled in the art, dormancy is one of two possible developmental states which mature seeds may show following desiccation and shedding (the other being germination). Embryo dormancy develops during late embryogenesis, and results in a lack of germination. Following imbibition of the mature shed seed it results in an inactive phase of plant growth during which development is deferred, although the embryo still maintains a high metabolic activity. Dormancy of mature imbibed seeds occurs even under environmental conditions that would favour germination, indicating that the process is not simply a lack of correct conditions. During dormancy, cells within the mature embryo are maintained in an arrested state, and nuclear DNA values obtained from A. fatua embryos indicate that the cell-cycle is held in G1 and DNA replication does not occur during imbibed dormancy (Elder and Osborne 1993). Dormancy is probably an evolutionary strategy that allows survival of seeds through adverse conditions, and dispersal of seed germination through time. Dormancy is therefore a very important phase of plant development required both for the inhibition of germination prior to completion of embryogenesis, and for the pre-germinative survival of mature seeds. It is also an important agronomic trait, with the market value of wheat being determined, inter alia, by its Hagberg Falling Number, which measures the degree to which some germination-related processes have progressed (discussed in relation to plant breeding hereinafter).
Seeds of the persistent weed A. fatua can show very high levels of embryo dormancy (Simpson 1978).
Embryos with primary dormancy go through a time and environment sensitive process of after-ripening in the dry seed, that is manifested by loss of dormancy in the imbibed seed (Mayer and Poljakoff-Mayber 1989). Dormancy can subsequently be reimposed on after-ripened dry embryos under specific environmental conditions (xe2x80x98inducedxe2x80x99 or xe2x80x98secondaryxe2x80x99 dormancy). These features indicate that signals perceived by the dry seed influence developmental choices following imbibition, resulting in either dormancy or germination (Hilhorst and Karssen 1992). Recent work on the water status of embryos of A. fatua has demonstrated that individual enzymatic and non-enzymatic reactions, rather than metabolic processes control this dormancy/non-dormancy switch in the dry seed (Foley 1994). Others have proposed that the process may involve kinase-phosphatase interactions (Trewavas 1987).
Many studies have analysed the genetic control of embryo dormancy (Hilhorst and Karssen 1992). Results obtained from experiments with inbred lines of A. fatua have suggested that in this species dormancy may be controlled by three loci, two that promote dormancy (L1 and L2), and one that promotes after-ripening (E) (Jana et al., 1979,
Jana et al. 1988). These dormancy genes have not been cloned or characterised, their existence was inferred from statistical analysis of segregation for dormancy phenotypes among the progeny from a cross between two different strains.
There is currently a requirement for materials and methods which have utility in the identification or molecular tagging of the genes responsible for PHS in wheat, or which could be used in the manipulation of the PHS trait in wheat or other plants.
Thus it can be seen that the provision of such materials or methods would provide a contribution to the art.
The present inventors have for the first time identified a gene from the oat Avena fatua which encodes a protein which has a high degree of similarity to known VP1/ABI 3 related transcription factors. The expression product of this gene has been termed afVP1.
By studying imbibed mature seeds, the expression of afVP1 has been correlated with the dormant phenotype (primary dormancy, secondary dormancy and after-ripening) in oat. In particular, the present inventors have demonstrated that wild oat has the potential for extremely high levels of dormancy in the mature dry seed, and that expression of afVP 1 is absolutely correlated to the dormant phenotype in imbibed mature seeds. This is the first demonstration of expression of a VP 1-homologue in a developmental situation other than embryogenesis. It indicates that afVP1 activity keeps mature seeds dormant, and inhibits germinationxe2x80x94it can thus be used to maintain or impose sufficient intensity and duration of dormancy to avoid PHS before harvest.
In addition to establishing an important role for afVP1 in the control of after-ripening and both primary and secondary dormancy. The present inventors have also employed afVP1 to identify the hitherto unobtained wheat VP1 homolog (hereinafter taVP1) and map its genomic position. As will be discussed in more detail below, the information made available by the present invention has important and industrially applicable implications for the detection and manipulation of PHS and other dormancy related traits in plants, and especially PHS in wheat. In particular work done by the inventors indicates that the ability to keep mature seeds dormant, and inhibit germination, has been lost by the wheat VP 1 due to breeding since domestication which has favoured the evolution of a crippled wheat VP 1 that cannot impose high levels of dormancy (resistance to PHS) on the mature seed.
Introduction of the wild oat afVP 1 into wheat can therefore be used to induce higher levels of dormancy (and thus resistance to PHS) in wheat as afVP 1 compensates for the crippled function of wheat VP 1.
Thus in a first aspect of the present invention there is provided a nucleic acid molecule, encoding afVP1, and having the sequence set out in Seq ID No 1 (shown in FIG. 4 (a)). A further afVP1 sequence, differing slightly from Seq ID No 1, has been deposited in the EMBL database under accession number AFJ001140 after the priority date of the present application.
The existence of an oat homologue to maize VP1 was reported briefly in a poster by M. J. Holdsworth xe2x80x9cDormancy-related expression of the wild oat (Avena fatua) homolog of the maize gene Viviparous 1 (Vp1)xe2x80x9d. Abstract, Poster No.49, Seventh International Symposium on Pre-Harvest Sprouting in Cereals 1995, Abashiri, Japan, July 1995.
Some comments about its properties were madexe2x80x94however the precise means of cloning the homolog, its sequence, and specific applications for it, were not disclosed.
Additionally Jones et al in April 1997 disclosed that afVP1 was correlated with primary and secondary dormancy (see Jones et al (1997) J Exp Bot 48 (Suppl) 45). Once again no details about how afVP1 could be obtained, or particular applications for it, were disclosed.
The nucleic acid molecules and their encoded polypeptide products (see below) according to the present invention may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of nucleic acid or genes of the species of interest or origin other than the sequence encoding a polypeptide with the required function. Nucleic acid according to the present invention may include cDNA, RNA, genomic DNA and may be wholly or partially synthetic.
The term xe2x80x9cisolatexe2x80x9d encompasses all these possibilities. Where a DNA sequence is specified, e.g. with reference to a figure, unless context requires otherwise the RNA equivalent, with U substituted for T where it occurs, is encompassed.
By virtue of its demonstrated properties, the nucleic acid of the first aspect may has utility, inter alia, in producing transformed crop plants having desirable primary or secondary dormancy, or after-ripening, properties, and in particular may be resistant to PHS.
In a further (second) aspect of the invention there is disclosed variants of the sequence provided, which may for instance be mutants or other derivatives, or naturally occurring alleles (or other homologues, including orthologues) of the sequence.
In the case of mutants and derivatives, the variant encodes a product which is homologous to the sequence of Seq ID No 1, and preferably which retains a functional characteristic that the product encoded by the variant has the afVP1 activity.
By xe2x80x98afVP1 activityxe2x80x99 is meant the ability to act as a transcription factor which is capable of activating some or preferably all of the genes which are activated by afVP1 (e.g. Em, C1) and repressing genes which are repressed by afVP1 (e.g. alpha-amylasexe2x80x94see Hoeker et al 1995 for maize VP1 activity in this regard). This can be assayed either directly using e.g. a reporter gene system linked to any of these genes or their promoters. Alternatively it may be assayed by preparing transformed plants and assaying its phenotypic effects in vivo (i.e. alteration of dormancy as described above).
Methodology for such transformation is described in more detail hereinafter.
Methods for producing such mutants or derivatives based on the sequence provided, and for identifying alleles (or other homologs) and then assessing homology are discussed below, and form one part of this aspect of the invention.
In all cases the nucleic acid molecule which is the mutant or other derivative is ultimately generated either directly or indirectly (e.g. via one or more amplification or replication steps) from oat afVP1 (including alleles thereof), preferably from a nucleic acid molecule comprising all or part of sequence ID No 1.
Changes to a sequence, to produce a mutant or derivative, may be by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide. Specifically included are parts or fragments (however produced) corresponding to portions of the sequences provided, and which encode polypeptides having afVP1 activity.
Changes may be desirable for a number of reasons, including introducing or removing the following features: restriction endonuclease sequences; other sites which are required for post translation modification; cleavage sites in the encoded polypeptide; motifs in the encoded polypeptide for post translational modification. All of these may assist in efficiently cloning and expressing an active polypeptide in recombinant form (as described below).
Other desirable mutation may be random or site directed mutagenesis in order to alter the activity (e.g. specificity) or stability of the encoded polypeptide. Particular regions of interest may be those which correspond to the regions of VP 1 which have been shown to function as either a transcriptional activation domain (amino acids 28-121, McCarty et al. 1991), or as a repressor domain (amino acids 238-375, Hoecker et al. 1995). Sections of these regions are highly conserved amongst all the VP 1 homologues (see FIG. 4, and discussion in the Examples) indicating that these sections may provide function to these regions. Comparison of the BR2 region, (shown previously to interact with other classes of transcription factors [Hill et al. 1996]), shows a high degree of similarity between afVP 1 and other homologues, indicating a conservation of function for this part of the protein. These comparisons (in terms of predicted amino acid structure) are shown in FIG. 4.
Those regions which differ from the corresponding parts of other VP1 s may also be of interest in that they may be responsible for the high dormancy demonstrated for oat.
Specifically embraced are derivatives which are truncated, or which have functional regions replaced with corresponding regions from other sources.
As is well-understood, homology at the amino acid level is generally in terms of amino acid similarity or identity.
Similarity allows for conservative variation, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. As is well known to those skilled in the art, altering the primary structure of a polypeptide (in this case the afVP1 protein) by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptides conformation.
Also included are homologs generated from afVP1 having non-conservative substitutions. As is well known to those skilled in the art, substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide""s three dimensional structure. In regions which are critical in determining the peptides conformation or activity such changes may confer advantageous properties on the polypeptide. Indeed, changes such as those described above may confer slightly advantageous properties on the peptide (e.g. altered stability, or DNA binding efficiency).
Similarity or homology (or identity, the terms are used synonymously) may be as defined and determined by the TBLASTN program, of Altschul et al. (1990) J. Mol. Biol. 215: 403-10, which is in standard use in the art, or, and this may be preferred, the standard program BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wis. USA, Wisconsin 53711) BestFit makes an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman.
Thus a mutant, derivative or allele (or other homolog) of the present invention shares homology with afVP1. Homology may be at the nucleotide sequence and/or amino acid sequence level. Preferably, the nucleic acid and/or encoded amino acid sequence shares homology with the coding sequence or the sequence encoded by the nucleotide sequence of Seq ID No 1, preferably at least about 50%, or 60%, or 70%, or 80% homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% homology.
Homology may be over the full-length of the relevant sequence shown herein, or may more preferably be over a contiguous sequence of about or greater than about 20, 25, 30, 33, 40, 50, 67, 133, 167, 200, 233, 267, 300, 333, 400, 450, 500, 550, 600 or more amino acids or codons, compared with the relevant amino acid sequence or nucleotide sequence as the case may be.
Thus a variant amino acid sequence in accordance with the present invention may include within the sequence shown in FIG. 4, a single amino acid change with respect to the sequence shown in FIG. 4, or 2, 3, 4, 5, 6, 7, 8, or 9 changes, about 10, 15, 20, 30, 40 or 50 changes, or greater than about 50, 60, 70, 80 or 90 changes. In addition to one or more changes within the afVP1 amino acid sequence shown in FIG. 4, a variant amino acid sequence may include additional amino acids at the C-terminus and/or N-terminus. Naturally, changes to the nucleic acid which make no difference to the encoded amino acid sequence (i.e. xe2x80x98degeneratively equivalentxe2x80x99) are included.
A further part of the present invention provides a method of identifying and cloning further afVP1 homologues or alleles from plant species which method employs a nucleotide sequence as described above.
As mentioned earlier, the present inventors have already used this method to identify a hitherto unidentified wheat VP1 homolog (termed taVP1)xe2x80x94this notwithstanding the failure of earlier workers (e.g. Cadle et al (1994)) who used maize VP1 to probe wheat. Nucleic acid molecules identified using this method form one part of the second aspect of the invention e.g. taVP1, as do mutants and derivatives of those genes.
The originally derived nucleotide sequences of various clones of taVP1 are shown in FIGS. 6 and 8.
Later sequences for the fully sequenced clone (designated taVP1xe2x80x94referred to as Seq ID No 2) and clones 2, 3, 4, 5, 6, and 9 are shown in FIGS. 10(a)-(g).
It is apparent the sequences shown that none of them encodes a full length protein i.e. they appear to be crippled to various degrees. This has important implications for improving dormancy in wheat, as regards molecular biology-based methods for transforming or breeding improved plants.
It may be noted that McKibbin et al, in a poster at SEB meeting in April 1997 reported using the wild oat homolog of VP1 in N-blots of wheat mRNA from various cultivars to demonstrate that expression levels of certain mRNAs were reduced in non-dormant phenotypes (see McKibbin et al (1997) J Exp Bot 48: (Suppl) 47). However no data regarding the sequence of the putative wheat (or oat) homolog(s) was presented.
When identifying homologs using the strategies below, if need be clones or fragments identified in the search can be extended to produce full length molecules. For instance if it is suspected that they are incomplete, the original DNA source (e.g. a clone library, poly(A)RNA extracted from embryos) can be revisited to isolate missing portions e.g. using sequences, probes or primers based on that portion which has already been obtained to identify other clones containing overlapping sequence.
In one embodiment, nucleotide sequence information provided herein may be used in a data-base (e.g. of ESTs) search to find homologous sequences, expression products of which can be tested for ability as described below.
In a further embodiment, a homolog or allele in accordance with the present invention is also obtainable by means of a method which includes:
(a) providing a preparation of nucleic acid, e.g. a genomic or cDNA library),
(b) providing a nucleic acid molecule having a nucleotide sequence shown in or complementary to a nucleotide sequence shown in Seq ID No 1 preferably from within the coding sequence (i.e. encoding for the afVP1 amino acid sequence shown in FIG. 4), most preferably the probe used is distinctive or characteristic of afVP1 rather than other, known, VP1 analogs,
(c) contacting nucleic acid in said preparation with said nucleic acid molecule under conditions for hybridisation of said nucleic acid molecule to any said gene or homologue in said preparation, and identifying said gene or homologue if present by its hybridisation with said nucleic acid molecule.
xe2x80x9cDistinctivexe2x80x9d or xe2x80x9ccharacteristicxe2x80x9d regions of afVP1 can be determined with reference to comparisons with know VP 1 homologues, for instance those shown in FIG. 4. Such regions and specific oligonucleotides are found away from the conserved regions which are described in more detail in Example 2 below, and will allow those skilled on the art to design probes and primers which will not hybridise with prior art sequences.
Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells.
Test nucleic acid may be provided from a cell as genomic DNA, cDNA or RNA, or a mixture of any of these, preferably as a library in a suitable vector. If genomic DNA is used the probe may be used to identify untranscribed regions of the gene (e.g. promoters etc.), such as is described hereinafter.
Preliminary experiments may be performed by hybridising under low stringency conditions. For probing, preferred conditions are those which are stringent enough for there to be a simple pattern with a small number of hybridisations identified as positive which can be investigated further.
It is well known in the art to increase stringency of hybridisation gradually until only a few positive clones remain. Suitable conditions would be achieved when a large number of hybridising fragments were obtained while the background hybridisation was low. Using these conditions nucleic acid libraries, e.g. cDNA libraries representative of expressed sequences, may be searched. Those skilled in the art are well able to employ suitable conditions of the desired stringency for selective hybridisation, taking into account factors such as oligonucleotide length and base composition, temperature and so on.
For instance, screening may initially be carried out under conditions, which comprise a temperature of about 37xc2x0 C. or less, a formamide concentration of less than about 50%, and a moderate to low salt (e.g. Standard Saline Citrate (xe2x80x98SSCxe2x80x99)=0.15 M sodium chloride; 0.15 M sodium citrate; pH 7) concentration.
Alternatively, a temperature of about 50xc2x0 C. or less and a high salt (e.g. xe2x80x98SSPExe2x80x99=0.180 mM sodium chloride; 9 mM disodium hydrogen phosphate; 9 mM sodium dihydrogen phosphate; 1 mM sodium EDTA; pH 7.4). Preferably the screening is carried out at about 37xc2x0 C., a formamide concentration of about 20%, and a salt concentration of about 5xc3x97SSC, or a temperature of about 50xc2x0 C. and a salt concentration of about 2xc3x97SSPE. These conditions will allow the identification of sequences which have a substantial degree of homology (similarity, identity) with the probe sequence, without requiring the perfect homology for the identification of a stable hybrid e.g. 70% homology.
Suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42xc2x0 C. in 0.25M Na2HPO4, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55xc2x0 C. in 0.1xc3x97SSC, 0.1% SDS. For detection of sequences that are greater than about 90% identical, suitable conditions include hybridization overnight at 65xc2x0 C. in 0.25M Na2HPO4, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60xc2x0 C. in 0.1xc3x97SSC, 0.1% SDS.
Binding of a probe to target nucleic acid (e.g. DNA) may be measured using any of a variety of techniques at the disposal of those skilled in the art. For instance, probes may be radioactively, fluorescently or enzymatically labelled. Other methods not employing labelling of probe include amplification using PCR (see below), RNxe2x80x2ase cleavage and allele specific oligonucleotide probing. The identification of successful hybridisation is followed by isolation of the nucleic acid which has hybridised, which may involve one or more steps of PCR or amplification of a vector in a suitable host.
In a further embodiment, hybridisation of nucleic acid molecule to an allele or homologue may be determined or identified indirectly, e.g. using a nucleic acid amplification reaction, particularly the polymerase chain reaction (PCR). PCR requires the use of two primers to specifically amplify target nucleic acid, so preferably two nucleic acid molecules with sequences characteristic of afVP1 are employed. However, if RACE is used (see below) only one such primer may be needed.
PCR techniques for the amplification of nucleic acid are described in U.S. Pat. No. 4,683,195 and Saiki et al.
Science 239: 487-491 (1988). References for the general use of PCR techniques include Mullis et al, Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR technology, Stockton Press, N.Y., 1989, Ehrlich et al, Science, 252:1643-1650, (1991), xe2x80x9cPCR protocols; A Guide to Methods and Applicationsxe2x80x9d, Eds. Innis et al, Academic 795 Press, New York, (1990).
Prior to any PCR that is to be performed, the complexity of a nucleic acid sample may be reduced where appropriate by creating a cDNA library for example using RT-PCR or by using the phenol emulsion reassociation technique (Clarke et al. (1992) NAR 20, 1289-1292) on a genomic library.
Thus a method involving use of PCR in obtaining nucleic acid according to the present invention may include:
(a) providing a preparation of plant nucleic acid,
(b) providing a pair of nucleic acid molecule primers useful in (i.e. suitable for) PCR, at least one said primers having a sequence shown in or complementary to a sequence shown in Seq ID No 1 as described in above,
(c) contacting nucleic acid in said preparation with said primers under conditions for performance of PCR,
(d) performing PCR and determining the presence or absence of an amplified PCR product. The presence of an amplified PCR product may indicate identification of a gene of interest or fragment thereof.
Thus the methods of the invention may include hybridisation of one or more (e.g. two) probes or primers to target nucleic acid. Where the nucleic acid is double-stranded DNA, hybridisation will generally be preceded by denaturation to produce single-stranded DNA. The hybridisation may be as part of a PCR procedure, or as part of a probing procedure not involving PCR. An example procedure would be a combination of PCR and low stringency hybridisation. A screening procedure, chosen from the many available to those skilled in the art, is used to identify successful hybridisation events and isolated hybridised nucleic acid.
An oligonucleotide for use in probing or PCR may be about 30 or fewer nucleotides in length (e.g. 18, 21 or 24). Generally specific (i.e. xe2x80x9cdistinctivexe2x80x9d or xe2x80x9ccharacteristicxe2x80x9d) primers are upwards of 14 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16-24 nucleotides in length may be preferred. Those skilled in the art are well versed in the design of primers for use processes such as PCR. If required, probing can be done with entire restriction fragments of the gene disclosed herein which may be 100""s or even 1000""s of nucleotides in length.
Some preferred oligonucleotides have a corresponding to bases 1 to 892 or 600 to 892 of Seq ID No 1. Primers may correspond to 1398 to 1417 or 2272 to 2290 of Seq ID No 1 (which is 1410-1429 and 2285-2302 on the afVP1 sequence deposisted on the EMBL database under accession number AFJ001140).
In a further (third) aspect of the present invention, the nucleic acid described above is in the form of a recombinant and preferably replicable vector.
xe2x80x9cVectorxe2x80x9d is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication).
Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in both the actinomycetes and related species and in bacteria and/or eucaryotic (e.g. higher plant, mammalian, yeast or fungal cells).
A vector including nucleic acid according to the present invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.
However, in a preferred embodiment, the nucleic acid in the vector is under the control of (operably linked to) an appropriate promoter or other regulatory elements for expression in a host cell such as a microbial, e.g. bacterial, or plant cell. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of CDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
By xe2x80x9cpromoterxe2x80x9d is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3xe2x80x2 direction on the sense strand of double-stranded DNA).
xe2x80x9cOperably linkedxe2x80x9d means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is xe2x80x9cunder transcriptional initiation regulationxe2x80x9d of the promoter.
Generally speaking, those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press.
Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis (see above), sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley and Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference. Specific procedures and vectors previously used with wide success upon plants are described by Bevan (Nucl. Acids Res. 12, 8711-8721 (1984)) and Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148).
If desired, selectable genetic markers may be used consisting of chimaeric genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate.
Thus this aspect of the invention provides a gene construct, preferably a replicable vector, comprising a promoter operatively linked to a nucleotide sequence provided by the present invention, such as the afVP1 gene shown in Seq ID No 1, a homologue thereof (e.g. taVP1), or any active mutant, derivative or allele thereof.
Suitable promoters include the Cauliflower Mosaic Virus 35S (CaMV 35S) gene promoter that is expressed at a high level in virtually all plant tissues (Benfey et al, 1990a and 1990b); the cauliflower meri 5 promoter that is expressed in the vegetative apical meristem as well as several well localised positions in the plant body, e.g. inner phloem, flower primordia, branching points in root and shoot (Medford, 1992; Medford et al, 1991) and the Arabidopsis thaliana LEAFY promoter that is expressed very early in flower development (Weigel et al, 1992).
Another strong promoter is the rice actin promoter. Also advantageous in the present context is the ubiquitin promoter which is expressed strongly in embryos (see Christenson and Quail (1996) Transgenic Research 5: 2133-2218.
Previous work in Arabidopsis has shown that constitutive expression of ABI 3 causes no negative effects on plant growth, and so expression of afVP 1 throughout a plant (e.g. wheat) may not have negative side effects on wheat plant growth.
However the promoter may include one or more sequence motifs or elements conferring developmental and/or tissue-specific regulatory control of expression. Other regulatory sequences may be included, for instance as identified by mutation or digest assay in an appropriate expression system or by sequence comparison with available information, e.g. using a computer to search on-line databases.
Thus in another embodiment of this aspect of the present invention, there is provided a gene construct, preferably a replicable vector, comprising an inducible promoter operably linked to a nucleotide sequence provided by the present invention, such as the afVP1 gene, a homolog from another plant species, e.g. a wheat taVP1, or any mutant, or allele thereof.
The term xe2x80x9cinduciblexe2x80x9d as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is xe2x80x9cswitched onxe2x80x9d or increased in response to an applied stimulus. The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. The preferable situation is where the level of expression increases upon application of the relevant stimulus by an amount effective to alter a phenotypic characteristic. Thus an inducible (or xe2x80x9cswitchablexe2x80x9d) promoter may be used which causes a basic level of expression in the absence of the stimulus which level is too low to bring about a desired phenotype (and may in fact be zero). Upon application of the stimulus, expression is increased (or switched on) to a level which brings about the desired phenotype.
A suitable inducible promoter is the GST-II-27 gene promoter which has been shown to be induced by certain chemical compounds which can be applied to growing plants. The promoter is functional in both monocotyledons and dicotyledons. It can therefore be used to control gene expression in a variety of genetically modified plants, including field crops such as canola, sunflower, tobacco, sugarbeet, cotton; cereals such as wheat, barley, rice, maize, sorghum; fruit such as tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, and melons; and vegetables such as carrot, lettuce, cabbage and onion. The GST-II-27 promoter is also suitable for use in a variety of tissues, including roots, leaves, stems and reproductive tissues.
Other advantageous promoters may be those which function at particular developmental stages (e.g. embryogenesis)xe2x80x94for instance the Em promoter, or the taVP1 wheat promoter which is discussed hereinafter.
In a fourth aspect the present invention also provides methods comprising introduction of such a construct into a plant cell and/or induction of expression of a construct within a plant cell, by application of a suitable stimulus, an effective exogenous inducer.
The vectors described above may be introduced into hosts by any appropriate method e.g. conjugation, mobilisation, transformation, transfection, transduction or electoporation.
However, when introducing a chosen gene construct into a cell, certain considerations must be taken into account, well known to those skilled in the art. The nucleic acid to be inserted should be assembled within a construct which contains effective regulatory elements which will drive transcription. There must be available a method of transporting the construct into the cell. Once the construct is within the cell membrane, integration into the endogenous chromosomal material either will or will not occur. Finally, as far as plants are concerned the target cell type must be such that cells can be regenerated into whole plants (see below).
Plants transformed with the DNA segment containing the sequence may be produced by standard techniques which are already known for the genetic manipulation of plants. DNA can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355, EP-A-0116718, NAR 12(22) 8711-87215 1984), particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al. (1987) Plant Tissue and Cell Culture, Academic Press), electroporation (EP 290395, WO 8706614 Gelvin Debeyserxe2x80x94see attached) other forms of direct DNA uptake (DE 4005152, WO 9012096, US 4684611), liposome mediated DNA uptake (e.g. Freeman et al. Plant Cell Physiol. 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNAS U.S.A. 87: 1228 (1990d) Physical methods for the transformation of plant cells are reviewed in Oard, 1991, Biotech. Adv. 9: 1-11.
Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Recently, there has been substantial progress towards the routine production of stable, fertile transgenic plants in almost all economically relevant monocot plants (Toriyama, et al. (1988) Bio/Technology 6, 1072-1074; Zhang, et al. (1988) Plant Cell Rep. 7, 379-384; Zhang, et al. (1988) Theor Appl Genet 76, 835-840; Shimamoto, et al. (1989) Nature 338, 274-276; Datta, et al. (1990) Bio/Technology 8, 736-740; Christou, et al. (1991) Bio/Technology 9, 957-962; Peng, et al. (1991) International Rice Research Institute, Manila, Philippines 563-574; Cao, et al. (1992) Plant Cell Rep. 11, 585-591; Li, et al. (1993) Plant Cell Rep. 12, 250-255; Rathore, et al. (1993) Plant Molecular Biology 21, 871-884; Fromm, et al. (1990) Bio/Technology 8, 833-839; Gordon-Kamm, et al. (1990) Plant Cell 2, 603-618; D""Halluin, et al. (1992) Plant Cell 4, 1495-1505; Walters, et al. (1992) Plant Molecular Biology 18, 189-200; Koziel, et al. (1993) Biotechnology 11, 194-200; Vasil, I. K. (1994) Plant Molecular Biology 25, 925-937;
Weeks, et al. (1993) Plant Physiology 102, 1077-1084; Somers, et al. (1992) Bio/Technology 10, 1589-1594; WO92/14828). In particular, Agrobacterium mediated transformation is now emerging also as an highly efficient alternative transformation method in monocots (Hiei et al. (1994) The Plant Journal 6, 271-282).
Particularly of interest on the present case is the fact that the generation of fertile transgenic plants has been achieved in the cereals rice, maize, wheat, oat, and barley (reviewed in Shimamoto, K. (1994) Current Opinion in Biotechnology 5, 158-162.; Vasil, et al. (1992) Bio/Technology 10, 667-674; Vain et al., 1995, Biotechnology Advances 13 (4): 653-671; Vasil, 1996, Nature Biotechnology 14 page 702).
Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium is inefficient or ineffective. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, eg bombardment with Agrobacterium coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233).
Following transformation, a plant may be regenerated, e.g. from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vasil et al., Cell Culture and Somatic Cell Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applications, Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.
The particular choice of a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practising the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.
Thus this aspect of the present invention includes a method of transforming a plant cell involving introduction of a vector comprising the afVP1 sequence (or a mutant or derivative thereof) into a plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce the sequence of nucleotides into the genome.
In a fifth aspect of the invention, there is disclosed a host cell containing nucleic acid or a vector according to the present invention, especially a plant or a microbial cell.
The invention further encompasses a host cell transformed with nucleic acid or a vector according to the present invention, especially a plant or a microbial cell, and most preferably a crop plant e.g. wheat. Within the cell, the nucleic acid may be incorporated within the chromosome. There may be more than one heterologous nucleotide sequence per haploid genome.
Thus in one embodiment of the invention there is provided a plant cell having incorporated into its genome nucleic acid, particularly heterologous nucleic acid, as provided by the present invention, under operative control of a regulatory sequence for control of expression. The coding sequence may be operably linked to one or more regulatory sequences which may be heterologous or foreign to the gene, such as not naturally associated with the gene for its expression. The nucleic acid according to the invention may be placed under the control of an externally inducible gene promoter to place expression under the control of the user.
The term xe2x80x9cheterologousxe2x80x9d is used broadly in this aspect to indicate that the gene/sequence of nucleotides in question have been introduced into said cells of the plant or an ancestor thereof, using genetic engineering, i.e. by human intervention. A heterologous gene (e.g. authentic afVP1) may replace an endogenous equivalent gene (e.g. taVP1; i.e. one which normally performs the same or a similar function) or the inserted sequence may be additional to the endogenous gene or other sequence. The heterologous (or exogenous or foreign) nucleic acid may be non-naturally occuring in cells of that type, variety or species. Thus the heterologous nucleic acid may comprise a coding sequence of or derived from a particular type of plant cell or species or variety of plant, placed within the context of a plant cell of a different type or species or variety of plant. A further possibility is for a nucleic acid sequence to be placed within a cell in which it or a homolog is found naturally, but wherein the nucleic acid sequence is linked and/or adjacent to nucleic acid which does not occur naturally within the cell, or cells of that type or species or variety of plant, such as operably linked to one or more regulatory sequences, such as a promoter sequence, for control of expression.
In the transgenic plant cell (i.e. transgenic for the nucleic acid in question) the transgene may be on an extra-genomic vector or incorporated, preferably stably, into the genome.
A plant may be regenerated from one or more transformed plant cells described above. Such plants form a sixth aspect of the invention.
A plant according to the present invention may be one which does not breed true in one or more properties. Plant varieties may be excluded, particularly registrable plant varieties according to Plant Breeders"" Rights. it is noted that a plant need not be considered a xe2x80x9cplant varietyxe2x80x9d simply because it contains stably within its genome a transgene, introduced into a cell of the plant or an ancestor thereof.
In addition to a plant, the present invention provides any clone of such a plant, seed, selfed or hybrid progeny is and descendants, and any part of any of these, such as cuttings, seed. The invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on. Also encompassed by the invention is a plant which is a sexually or asexually propagated off-spring, clone or descendant of such a plant, or any part or propagule of said plant, off-spring, clone or descendant.
Particularly embraced are the seeds or grains of a transformed plant as described above, and also products (e.g. human and animal foodstuffs) derived from or containing such seeds or grains.
In a seventh aspect, the invention provides a method of influencing or affecting the dormancy characteristics of a plant, preferably the viviparous or PHS phenotype of a plant, including the step of causing or allowing expression of a heterologous nucleic acid sequence, as discussed in relation to the first and second aspects of the invention, within cells of the plant.
This aspect particularly provides a method of including expression from nucleic acid Seq ID No 1 or 2, or a mutant, allele or derivative of those sequences, within cells of a plant (thereby producing the encoded polypeptide), following an earlier step of introduction of the nucleic acid into a cell of the plant or an ancestor thereof. The method may employ the vectors of the third aspect.
In the present invention, expression (or over-expression or endogenous sequences) may be achieved by introduction of the nucleotide sequence in a xe2x80x9csensexe2x80x9d orientation. Thus, the present invention provides a method of the method including causing or allowing expression of the product (polypeptide or nucleic acid transcript) encoded by heterologous nucleic acid according to the invention from that nucleic acid within cells of the plant.
The complete sequence corresponding to the coding sequence of afVP1 need not be used. For example fragments (i.e. active derivatives or mutants) of sufficient length may be used.
The sequence employed may be about 500 nucleotides or less, possibly about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, or about 100 nucleotides. It may be possible to use oligonucleotides of much shorter lengths, 14-23 nucleotides, although longer fragments, and generally even longer than about 500 nucleotides are preferable where possible, such as longer than about 600 nucleotides, than about 700 nucleotides, than about 800 nucleotides, than about 1000 nucleotides or more.
Down-regulation of expression of a target gene (e.g. a homolog identified in accordance with the second aspect of the invention such as taVP1xe2x80x94Seq ID No 2) may be achieved using anti-sense technology or xe2x80x9csense regulationxe2x80x9d (xe2x80x9cco-suppressionxe2x80x9d).
For instance, if it is desired to suppress dormancy (i.e. enhance PHS or xe2x80x98maltingxe2x80x99) then the nucleic acids of the present invention (e.g. taVP1, afVP1, other derivatives) may be used for this purpose in accordance with standard procedures for anti-sense or sense suppression.
In using anti-sense genes or partial gene sequences to down-regulate gene expression, a nucleotide sequence is placed under the control of a promoter in a xe2x80x9creverse orientationxe2x80x9d such that transcription yields RNA which is complementary to normal mRNA transcribed from the xe2x80x9csensexe2x80x9d strand of the target gene. See, for example, Rothstein et al, 1987; Smith et al, (1988) Nature 334, 724-726; Zhang et al, (1992) The Plant Cell 4, 1575-1588, English et al., (1996) The Plant Cell 8, 179-188. Antisense technology is also reviewed in Bourque, (1995), Plant Science 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496.
An alternative is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression. See, for example, van der Krol et al., (1990) The Plant Cell 2, 291-299; Napoli et al., (1990) The Plant Cell 2, 279-289; Zhang et al., (1992) The Plant Cell 4, 1575-1588, and U. S. Pat. No. 5,231,020. Recent work indicates that foreign (non-endogenous) homologous sequences may be particularly effective at inducing gene silencing in targeted endogenous genes. See e.g. Matzke, M. A. and Matzke, A. J. M. (1995), Trends in Genetics, 11: 1-3). This sequence homology may involve promoter regions or coding regions of the silenced gene (Matzke, M. A. and Matzke, A. J. M. (1993) Annu. Rev. Plant Physiol. Plant Mol. Biol., 44: 53-76, Vaucheret, H. (1993) C. R. Acad. Sci. Paris, 316: 1471-1483, Vaucheret, H. (1994), C. R. Acad. Sci. Paris, 317: 310-323, Baulcombe, D. C. and English, J. J. (1996), Current Opinion In Biotechnology, 7: 173-180, Park, Y-D., et al (1996), Plant J., 9: 183-194.
Thus the sequences of the present invention may have utility when used in plant species different to those from which they were derived (e.g. barley). In an eight aspect, the present invention also encompasses the expression product of any of the nucleic acid sequences disclosed, particularly those of the first and second aspects, and methods of making the expression product by expression from encoding nucleic acid therefore under suitable conditions, which may be in suitable host cells.
Following expression, the product may be isolated from the expression system (e.g. microbial) and may be used as desired, for instance in formulation of a composition including at least one additional component.
Alternatively (and indeed preferably) the product may perform its function in vivo, in this context the function being to influence the dormancy characteristics of a plant, preferably the viviparous or PHS phenotype of a plant.
In an ninth aspect, purified or semi-purified afVP1 or taVP1 protein, or a fragment, mutant, derivative or other variant thereof, e.g. produced recombinantly by expression from encoding nucleic acid therefor, may be used to raise antibodies employing techniques which are standard in the art. Antibodies and polypeptides comprising antigen-binding fragments of antibodies may be used in identifying homologs from other species as discussed further below, and also in labelling proteins.
Methods of producing antibodies include immunising a mammal (e.g. human, mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and might be screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al, 1992, Nature 357: 80-82). Antibodies may be polyclonal or monoclonal. As an alternative or supplement to immunising a mammal, antibodies with appropriate binding specificity may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.
Antibodies may have utility in testing for endogenous VP1-analog expression (especially taVP1 expression in wheat) as part of a dormancy/PHS assessment, as is discussed hereinafter.
Antibodies raised to a polypeptide or peptide can be used in the identification and/or isolation of homologous polypeptides, and then their encoding genes.
Thus, the present invention provides a method of identifying or isolating a polypeptide having one or more afVP1 (or taVP1 ) epitopes comprising screening candidate polypeptides with a polypeptide comprising the antigen-binding domain of an antibody (for example whole antibody or a fragment thereof) which is able to bind afVP1 (or taVP1 ) or a fragment or a derivative thereof or preferably has binding specificity for such a polypeptide.
Thus specific binding members such as antibodies and polypeptides comprising antigen binding domains of antibodies that bind and are preferably specific for afVP1 or taVP1 polypeptides or mutants or derivatives thereof represent part of the present invention, as do their use and methods which employ them.
Candidate polypeptides for screening may for instance be the products of an expression library created using nucleic acid derived from an plant of interest, or may be the product of a purification process from a natural source. A polypeptide found to bind the antibody may be isolated and then may be subject to amino acid sequencing. Any suitable technique may be used to sequence the polypeptide either wholly or partially (for instance a fragment of the polypeptide may be sequenced). Amino acid sequence information may be used in obtaining nucleic acid encoding the polypeptide, for instance by designing one or more oligonucleotides (e.g. a degenerate pool of oligonucleotides) for use as probes or primers in hybridization to candidate nucleic acid, or by searching computer sequence databases, as discussed further below.
The above description has generally been concerned with the coding parts of the afVP1 gene, and uses therefor. Also embraced within the present invention are untranscribed parts of that gene, or the taVP1 gene.
Thus a tenth aspect of the invention is a nucleic acid molecule encoding the promoter of the afVP1 gene or the taVP1 gene.
The promoter region may be readily identified using a probe or primer based on Seq ID No 1 or Seq ID no 2, as described in relation to earlier aspects. This can be used in the identification and isolation of a promoter from a genomic library containing DNA derived from a plant source. Techniques and conditions for such probing are well known in the art as is discussed above. Following probing, promoter activity is assessed using a test transcription system.
xe2x80x9cPromoter activityxe2x80x9d is used to refer to ability to initiate transcription. The level of promoter activity is quantifiable for instance by assessment of the amount of mRNA produced by transcription from the promoter or by assessment of the amount of protein product produced by translation of mRNA produced by transcription from the promoter. The amount of a specific mRNA present in an expression system may be determined for example using specific oligonucleotides which are able to hybridise with the mRNA and which are labelled or may be used in a specific amplification reaction such as the polymerase chain reaction.
Use of a reporter gene facilitates determination of promoter activity by reference to protein production. The reporter gene preferably encodes an enzyme which catalyses a reaction which produces a detectable signal, preferably a visually detectable signal, such as a coloured product. Many examples are known, including xcex2-galactosidase and luciferase. xcex2-galactosidase activity may be assayed by production of blue colour on substrate, the assay being by eye or by use of a spectro-photometer to measure absorbance. Fluorescence, for example that produced as a result of luciferase activity, may be quantitated using a spectrophotometer. Radioactive assays may be used, for instance using chloramphenicol acetyltransferase, which may also be used in non-radioactive assays. The presence and/or amount of gene product resulting from expression from the reporter gene may be determined using a molecule able to bind the product, such as an antibody or fragment thereof. The binding molecule may be labelled directly or indirectly using any standard technique.
Those skilled in the art are well aware of a multitude of possible reporter genes and assay techniques which may be used to determine promoter activity. Any suitable reporter/assay may be used and it should be appreciated that no particular choice is essential to or a limitation of the present invention.
Also embraced by the present invention is a promoter which is a mutant, derivative, or other homolog of the promoter identified as above. These can be generated or identified in similar manner to the derivatives discussed in the second aspect; they will share homology with the taVP1 or afVP1 promoters and retain promoter activity.
To find minimal elements or motifs responsible for tissue and/or developmental regulation, restriction enzyme or nucleases may be used to digest a nucleic acid molecule, or mutagenesis may be employed, followed by an appropriate assay (for example using a reporter gene such as luciferase) to determine the sequence required. Nucleic acid comprising these elements or motifs forms one part of the present invention.
In an eleventh aspect of the invention there is provided a nucleic acid construct, preferably an expression vector, including a promoter region or fragment, mutant, derivative or other homolog or variant thereof able to promote transcription as discussed above, operably linked to a heterologous gene, e.g. a coding sequence, which is preferably not the coding sequence with which the promoter is operably linked in nature.
The above aspects of the invention are concerned generally with methods and materials which have utility, inter alia, in manipulating PHS and/or other dormancy related traits (secondary and after-ripening) in crops, particularly wheat, by means of transformation.
However the identification of the wheat homolog taVP1 by the present inventors has also opened up the possibility of improved methods for generating plants having desirable characteristics as regards PHS and/or other dormancy related traits. These methods have as their basis the identification and molecular tagging of the taVP1 gene (using taVP1 cDNA as a probe in genomic southern blots) which has been achieved by the present inventors, as described below.
A plant breeding approach to improving the PHS properties of wheat has a number of advantages over transformation approaches, particularly as regards consumer confidence in the improved products and ease of regulatory approval.
Essentially, plant breeding is the process of bringing together new combinations of genes, from different parents, allowing them to reassort into recombinant genotypes carrying various mixtures of the original parental genes, then selecting individual progenies which carry genetic combinations superior to the original parents.
Dormancy is one of a number of characteristics described as xe2x80x9cquantitative traitsxe2x80x9d (i.e. varies over a continuous range as opposed to a trait such as grain colour which is an xe2x80x9call or nothingxe2x80x9d, discontinuous character).
Quantitative traits (=QTs) are controlled by several/many genes (situated on the chromosomes at several/many Quantitative Trait Loci=QTLs); and by xe2x80x9cenvironmentalxe2x80x9d variables including weather effects and experimental uncertainties of measurement. Breeding for desirable QTs thus demands the ability to effectively discriminate between genetic variants at a large number of QTLs, conventionally by using statistical techniques based on large samples and repeated trials.
Dormancy is a particularly difficult QT for the plant breeder because the effects of xe2x80x9cerrorxe2x80x9d variables are comparatively large, the trait itself is lost gradually during after-ripening, and experimental methods for testing dormancy are time/labour/material intensive. Since dormancy tests have to be carried out between one harvest and the next sowing (sometimes just a month or so), only limited time is available for empirical testing; for this reason wheat breeders usually defer dormancy testing until the later stages of a breeding programme when only a limited number of xe2x80x9celitexe2x80x9d progenies have passed through earlier rounds of selection for other, more easily selectable characters. This has the effect of reducing the amount of control which the breeder can exercise over the genetic combinations of dormancy QTLs passing into his new varieties.
When selecting progeny, rather than use xe2x80x9cdirectxe2x80x9d selection for dormancy (i.e. by measuring the trait itself), in which genetic combinations are unconsciously chosen on the basis of their statistical performance in empirical tests, it is possible to exploit xe2x80x9cmarker aidedxe2x80x9d selection, choosing progenies on the basis of discontinuous traits, each of which is simply controlled by genetic variants at a single (or small number) of genetic locus/loci. The conventional marker for dormancy in wheat is grain colour; it has been known for many years that red-seeded wheats tend to be more dormant than white-seeded wheats, and that grain colour is determined by dominant xe2x80x9credxe2x80x9d versus recessive xe2x80x9cwhitexe2x80x9d alleles (gene variants) at three major gene loci. By discarding any white-seeded progenies (detected by visual inspection after steeping grains in dilute aqueous sodium hydroxide) wheat breeders aim to eliminate the undesirable lack of dormancy associated with this character.
Thus one approach to breeding for PHS resistance is disclosed by Derera NF (1989) in xe2x80x9cBreeding for pre-harvest sprouting tolerancexe2x80x9d. pp111-128 in: NF Derera (Ed), xe2x80x9cPre-Harvest Field Sprouting in Cereals. CRC Press Inc.xe2x80x9d This gives some specific examples of breeding programmes which have been undertaken to produce new varieties with improved dormancy, including exploitation of red grain colour, breeding for dormancy in white-grained wheats, screening wheat genotypes for use as donors of dormancy genes in breeding, and a suggested scheme for dormancy breeding. The use of the Hagberg falling number test (to assess the viscosity of ground kernel material under moist and dry conditions) is also discussed as being a useful measure of sprouting resistance.
Another discussion of PHS is found in Mares D J (1989) xe2x80x9cPre-harvest sprouting damage and sprouting tolerance: assay methods and instrumentation.xe2x80x9d pp129-170 in the same volume as Derera (supra). This disclosed various methods used to select against sprouting. The Hagberg test, and its approval by various standardisation bodies, is also discussed. It is noted that an absence of sprouting damage (indicative of low PHS susceptibility) leads to high falling numbers ( greater than 400, generally 450-550).
The role of grain colour in dormancy, and its relationship with VP1, has been discussed in number of prior art papers. For instance the poster by Wilson (described in the prior art section above) discussed the possible orthology of the Red grain locus in wheat to the maize VP-1 locus. Similarly Sorrells and Wilson (1997) Crop Science 37: 691-697 discusses the relationship between maize red pericarp colour (controlled by the Pi gene) nd
VP1, and suggests that a (postulated) wheat VP1 homolog may express via a P homolog.
Unfortunately the association between grain redness and dormancy is not hard and fast: breeders of white-grained wheats (eg. Australia) or amber wheats (eg. durum) must resort to other QTLs, also it has been noted that red wheats vary widely in dormancy. The most dormant white wheats can be as dormant as the least dormant reds: within both colour groups there is a continuous spectrum of dormancy, the scores of red wheats are shifted to the more dormant end of the spectrum, but there is considerable overlap between the two groups. This makes it clear that grain colour is not the sole determinant of dormancy.
Thus it can be seen that further markers to assist in the assessment of PHS resistance and other dormancy-related traits would be beneficial in breeding improved cultivars.
Ideally such markers should be in as close genetic linkage with the QTL as possible, or even better that it have a direct effect on the QT (reducing or even better eliminating the possibility of recombination). Selecting particular alleles which are known to directly exert particular phentoypic effects is termed Direct Allele Selection (xe2x80x98DASxe2x80x99). Additionally the QTL which is marked should be an important determinant of the QT score. Finally it should also be practicable. Molecular markers, which depend on variation (polymorphisms) in the sequences of bases, are particularly useful in this regard.
The present inventors have now mapped the chromosomal location of the novel taVp1 genes which they isolated.
Briefly, an RFLP polymorphism was identified between two parents of an F2 mapping population, then the alleles present in each individual of the population were determined. These genotypes at the taVp1 locus were then compared with the genotypes of the same individuals at other, genetically linked loci determined previously (Devos et al 1992). Two of the three taVp1 loci were mapped in this way (one on chromosome 3A, one on 3D); the third locus was detected on chromosome 3B by nullisomic analysis.
As with the grain colour genes, taVp1 copies reside at three loci on the long arms of chromosomes 3A, 3B and 3D respectively. The results were compared to the consensus wheat map of Gale et al 1995 [(M. D. Gale, M. D. Atkinson, C. N. Chinoy, R. L. Harcourt, J. Jia, Q. Y. Li and K. M. Devos. 1995. Genetic maps of hexaploid wheat. pp29-40 in: Proceedings 8th International Wheat Genetics Symposium, Eds Z. S. Li and Z. Y. Xin, China Agricultural Scientech Press, Beijing] and homologous taVp1 loci were assigned to the interval between loci Xwg110 and Xpsr549 on the consensus map (see FIG. 7).
Interestingly it was found that the taVp1 genes are linked to the R (colour genes) only at a distance of about 25 centiMorgans. This is clear evidence that taVp1 and R genes are different, so that both marker systems can be used for manipulating dormancy, either in concert or independently. Both markers have direct effects, so loss of effect due to recombination is not a problem. The linkage interval between the two loci on each chromosome is large enough to allow a practicably high frequency of recovery of taVp1/R recombinants should these be required (eg. any existing linkage between a xe2x80x9cgoodxe2x80x9d taVp1 allele and a xe2x80x9credxe2x80x9d colour allele could realistically be broken for introduction of the taVp1-based dormancy into white wheats).
Apart from the option for breeding dormant white/amber wheats, taVp1 offers advantages over use of the colour marker arising from the availability of clones of wheat alleles. The problems with the colour marker are that it cannot be scored until the grain is ripe, and that no information is available about which of the R genes is present without lengthy and large-scale breeding experiments. Knowledge of the taVp1 DNA, RNA, and/or protein sequences allows the identification of individual alleles present in a sample of tissue, e.g. DNA from the first seedling leaf, or even taken from a seed prior to sowing.
Polymorphisms can be manifest in a number of ways. Structurally they will alter the characteristics of the DNA to bind probes and primers at particular sites, or its properties as a substrate for restriction analysis. Functionally they may affect the quality or quantity of mRNA or protein product which derives from the DNA. Thus, for instance, the presence of absence of a lesion in a promoter or other regulatory sequence may be assessed by determining the level of mRNA production by transcription or the level of polypeptide production by translation from the mRNA. The level of mRNA or protein will be affected not only by its rate of production, but also by its stability and rate of degradation.
Thus the sequence information (nucleic and/or protein product) disclosed herein enables the use of specific amplification, probing or other techniques, to carry out allele identification and hence germplasm classification.
xe2x80x9cNucleic acid sequencexe2x80x9d in this context embraces the coding sequences, introns, and promoters of the relevant allele, plus also post-transcriptional modifications of RNA. Thus tests may be carried out on preparations containing genomic DNA, cDNA and/or mRNA. Testing cDNA or mRNA has the advantage of the complexity of the nucleic acid being reduced by the absence of intron sequences, but the possible disadvantage of extra time and effort being required in making the preparations. RNA may be more difficult to manipulate than DNA because of the wide-spread occurrence of RNxe2x80x2ases.
xe2x80x9cProtein sequencexe2x80x9d in this context covers both the primary structure, plus post-translational protein modifications. Under certain circumstances the total absence of a detectable protein product will be indicative of alterations in the encoded protein sequence.
Generally the methods may make use of biological samples from one or more plants or cells (e.g. in a seed)that are suspected to contain the nucleic acid sequences or polypeptide.
The following method are exemplary only. Those skilled in the art will appreciate that other methods which may be devised without burden on the basis of the information made available by the present inventors also form part of the present invention. For instance a number of methods for determining the presence and identity of polymorphic molecular markers (in the context of biodiversity analysis) are disclosed by Karp et al (1997) Biotechnology 15: 625-628. Such methods may have analagous utility in carrying out the present invention.
1) At the nucleic acid level, identification may involve hybridisation of a suitable specific oligo- or poly-nucleotide probe, such as a fragment of those disclosed herein, or further allelic sequences established using the information disclosed herein. Where the nucleic acid target is double-stranded DNA, hybridisation will generally be preceded by denaturation to produce single-stranded DNA. Such methods include Southern and Northern hybridisations, which can be used both qualitatively or quantitively (e.g. to assess mRNA level). A screening procedure, chosen from the many available to those skilled in the art, may be used to identify successful hybridisation events and isolate hybridised nucleic acid. For instance, probes may be radioactively, fluorescently or enzymatically labelled.
Preferably the screening is carried out with a variantxe2x80x94or allele-specific probexe2x80x94this is particular useful for DAS. Such a probe corresponds in sequence to a region of the gene, or its complement, containing a sequence alteration known to be associated with the trait of interest. Under suitably stringent conditions, specific hybridisation of such a probe to test nucleic acid is indicative of the presence of the sequence alteration in the test nucleic acid. For efficient screening purposes, more than one probe may be used on the same test sample.
When screening for particular alleles, the nucleic acid in the sample may initially be amplified, e.g. using PCR, to increase the amount of the analyte as compared to other sequences present in the sample. This allows the target sequences to be detected with a high degree of sensitivity if they are present in the sample. This initial step may be avoided by using highly sensitive array techniques
Approaches which rely on hybridisation between a probe and test nucleic acid and subsequent detection of a mismatch may be employed. Under appropriate conditions (temperature, pH etc.), an oligonucleotide probe will hybridise with a sequence which is not entirely complementary. The degree of base-pairing between the two molecules will be sufficient for them to anneal despite a mis-match. Various approaches are well known in the art for detecting the presence of a mis-match between two annealing nucleic acid molecules. For instance, RNxe2x80x2ase A cleaves at the site of a mis-match. Cleavage can be detected by electrophoresing test nucleic acid to which the relevant probe or probe has annealed and looking for smaller molecules (i.e. molecules with higher electrophoretic mobility) than the full length probe/test hybrid. Other approaches rely on the use of enzymes such as resolvases or endonucleases. Thus, an oligonucleotide probe that has the sequence of a region of the normal gene (either sense or anti-sense strand) in which polymorphisms associated with the trait of interest are known to occur may be annealed to test nucleic acid and the presence or absence of a mis-match determined. Detection of the presence of a mis-match may indicate the presence in the test nucleic acid of a mutation associated with the trait. On the other hand, an oligonucleotide probe that has the sequence of a region of the gene including a mutation associated with disease resistance may be annealed to test nucleic acid and the presence or absence of a mis-match determined. The presence of a mis-match may indicate that the nucleic acid in the test sample has the normal sequence, or a different mutant or allele sequence. In either case, a battery of probes to different regions of the gene may be employed.
(ii) Allele- or variant- (or even genome-) specific oligonucleotides may similarly be used in PCR to specifically amplify particular sequences if present in a test sample. Assessment of whether a PCR band contains a gene variant may be carried out in a number of ways familiar to those skilled in the art.
The PCR product may for instance be treated in a way that enables one to display the mutation or polymorphism on a denaturing polyacrylamide DNA sequencing gel, with specific bands that are linked to the gene variants being selected. It may also be desirable to analyse DNA fragment size, restriction site variation (e.g. CAPSxe2x80x94cleaved amplified polymorphic sites) and so on. Sequence Tagged Site (STS) Polymerase Chain Reaction (PCR) is rapid, specific, and does not require use of radiosotopes/autoradiography.
By way of Examples, the following primers could be used to distinguish the certain alleles disclosed in FIGS. 6 and 8. Further primer combinations can be devised without burden by those skilled in the art for new taVP1 alleles if and when they are identified (for instance by use of the materials and methods disclosed herein).
Non specific primer (reverse compliment):
Position 2210/clone 10: CGT CAC ATC TGA CCG ATA GC
Primers to differentiate clones 6+9 from 5+10:
6+9 specific: position 1696: CAT CTC AGG TGT GGA GCA TGC
5+10 specific: position 1691: CGG CAC ATC TCA GAT TTT GGC CC
Primers to differentiate clones 5+6 from 9+10:
5+6 specific: position 1432: GCG GCA GCA GGG TGC GAG G
9+10 specific: position 1432: GCG GCA GCA GGT GCA TGC ATG
Further primers which are specific for the A- B- and D-genomes are discussed in the Examples hereinafter.
(iii) RFLP, hybridized to homologous or heterologous probes based on the sequences disclosed herein. The presence of differences in sequence of nucleic acid molecules may be detected by means of restriction enzyme digestion, such as in a method of DNA fingerprinting where the restriction pattern produced when one or more restriction enzymes (chosen on the basis of the sequences disclosed herein) are used to cut a sample of nucleic acid is compared with the pattern obtained when a sample containing the desired allele or a variant digested with the same enzyme or enzymes. Amplified Fragment Length Polymorphism (AFLP) can be carried out using primers devised on the basis of the sequences disclosed herein. The strength of AFLP is the ability to screen a large number of different marker molecules in a single test. Analysis of the products can be carried out using e.g. by gel electrophoresis, capillary electrophoresis.
(iv) RT-PCR is based on the amplification of RNA, which may be qualitative or quantitative. Essentially this method uses reverse transcriptase to generate a DNA copy of plant mRNA.
(v) Variations in antibody/antigen complexes between plant protein extracts and antibodies targeted to protein epitopes. This is particular useful for distinguishing functional from non-functional proteins (e.g. truncated forms, wherein a particular allele contains a premature stop codon). Thus a sample may be tested for the presence of a binding partner for an antibody (or mixture of antibodies), specific for one or more allelic variants. Binding is assessed by any means commonly known to those skilled in the art. Where a panel of antibodies is used, different reporting labels may be employed for each antibody so that binding of each can be determined.
A specific binding member such as an antibody may be used to isolate and/or purify its binding partner polypeptide from a test sample, to allow for sequence and/or biochemical analysis of the polypeptide to determine whether it has the sequence and/or properties of the wild-type polypeptide or a particular mutant, variant or allele thereof. Amino acid sequence is routine in the art using automated sequencing machines.
Thus the work of the present inventors offers a completely novel approach for manipulating seed dormancy in breeding programmes, exploiting the taVp1 gene sequence which was previously unknown for direct selection of alleles controlling a QT for which currently available techniques are laborious, slow, and inefficient.
Various aspects of the invention will now be discussed in more detail:
In one aspect of the invention there is disclosed a method for assessing the PHS and/or other dormancy related properties of a wheat plant, the method comprising use of the molecular marker taVP1 which occurs in the interval between loci Xwg110 and Xpsr549 on the wheat group 3 consensus map.
In a further aspect of the invention these is disclosed of producing a cultivar comprising the steps of selecting a parent line having desired PHS and/or other dormancy related properties, breeding with that line, and selecting progeny on the basis of the molecular marker taVP1 described above.
Preferably the selection of the parent line(s) and/or progeny is done on the basis of specific superior alleles (i.e. DAS). This allows precise manipulation of variation of PHS via selection of progenies with appropriate, desired functional activity of characterised alleles. Thus PHS may be improved by selection for high levels of expression of fully-functional alleles, firstly by selecting parents carrying desirable genomic copies of taVp1 gene(s), then by selecting progeny which express these alleles strongly at appropriate stages of seed development and maturation.
The assessment can be on the basis of analysing taVP1 DNA, RNA or protein as described above, and then correlating the result of the analysis with the expected PHS phenotype.
Parent plants possessing favoured alleles may be obtained from within an existing variety genepool, or prepared mutants from within an elite genepool.
Alternatively desirable alleles or may also be detected and transferred from one or more of the many wild or cultivated relatives of the plant, for which established methods are available for the introduction of xe2x80x9calienxe2x80x9d variation into the plant genome. The correlation of the PHS trait with the afVP1 sequence greatly facilitates the identification and selection of desirable alleles in exotic germplasm. This is especially useful for species in which genetic variation in cultivated germplasm is limited (e.g. wheat T aestivumxe2x80x94see Chao et al (1989) Theor Appl Genet 88:717-721).
In the past, using traditional methods, problems with using exotic germplasm have included the low frequency of desirable alleles, and difficulties with linkage drag and polygenic inheritance. Such problems will be minimised by use of the present invention.
Lines may be produced by breeding from selected lines in accordance with standard techniques well known to those skilled in the art.
Clearly the PHS phenotype can be manipulated up or down; for applications in which dormancy is undesirable, e.g. malting, the same information and techniques could be employed to select in the reverse direction, i.e. to fix defective or poorly expressed copies of taVP1.
The demonstration by the present inventors that the afVP1 and seed colour gene alleles are linked but separate demonstrates that both marker systems can be used for manipulating dormancy, either in concert or independently. Thus methods of selection based on taVP1 alone (e.g. in white/amber grained wheats) or both taVP1 and red grain colour (e.g. in red wheats) also form part of the present invention.
Methods in which the taVP1 allele in particular plant or line is assessed and the result of the assessment is correlated directly with expected PHS phenotype, e.g. for the purposes of timing harvest, form a further part of the invention.
The taVP1 assessment may also be used to assess pedigree or phylogenetic origin if desired.
Nucleic acid-based determination of the identity of a particular taVP1 allele (e.g. as in the methods described above) may be combined with determination of the genotype of the flanking linked genomic DNA and other unlinked genomic DNA using established sets of markers such as RFLPs, microsatellites or SSRS, AFLPS, RAPDs etc. This enables the researcher or plant breeder to select for not only the presence of the desirable taVP1 allele but also for individual plant or families of plants which have the most desirable combinations of linked and unlinked genetic background. Such recombinations of desirable material may occur only rarely within a given segregating breeding population or backcross progeny. Direct assay of the taVP1 locus as afforded by the present invention allows the researcher to make a stepwise approach to fixing (making homozygous) the desired combination of flanking markers and taVP1 alleles, by first identifying individuals fixed for one flanking marker and then identifying progeny fixed on the other side of the locus all the time knowing with confidence that the desirable taVP1 allele is still present.
The sequence information provided herein also allows the design of diagnostic tests and kits for determination of the presence of particular taVP1 and afVP1 alleles, in any given plant, cultivar, variety, population, landrace, part of a family or other selection in a breeding programme or other such genotype. A diagnostic test may be based on determination of the presence or absence of a particular allele by means of nucleic acid or polypeptide determination.
Plants which are generated (or assessed and or approved) using the taVP1-allele assessment methods of the present invention form a further aspect of the invention. Plants in this context embraces cultivars, and seeds, microspores, protoplasts, cotyledons, zygotes (ovules, pollen) and vegetative parts derived therefrom. It further embraces any clone of such a plant, selfed or hybrid progeny and descendants, and any part of any of these, such as cuttings and seed. Products made from such plants e.g. milled or malted grains, flour etc. are also embraced by the invention.
The invention will now be further illustrated with reference to the following non-limiting Figures and Examples. Other embodiments falling within the scope of the invention will occur to those skilled in the art in the light of these.
Seq ID No 1: (see FIG. 4(a)) xe2x80x9cafVP1xe2x80x9d CDNA sequence.
Seq ID No 2: (See FIG. 10(a)): xe2x80x9ctaVP1xe2x80x9d CDNA sequence.